EP1792980B1 - Collagen biomatrix and methods to poduce it - Google Patents

Abstract

Cartilage replacement (A) prepared by (i) incorporating dedifferentiated cartilage cells (B) into a three-dimensional, gel-like biomatrix containing at least 1.5 mg/ml collagen in buffered, serum-containing cell culture medium and (ii) culturing (B) so that they redifferentiate and/or replicate to a form a transplantable (A). Independent claims are also included for the following: (a) similar material in which the biomatrix is seeded with differentiated (B) and these cultured for replication, while remaining differentiated; (b) similar material in which (B) are checked for function, morphology and/or differentiation status simultaneously with production of (A) or afterwards; and (c) biomatrix for culturing cells, particularly (B).

Description

The invention relates to a biomatrix and to processes for their preparation.

Joint disease is a widespread disease associated with a variety of distress for the individuals involved. For example, bone and cartilage defects play a central role in the pathogenesis of osteoarthritis, but also in post-traumatic conditions and erosive endoprostheses. Such defects can be treated by using autologous or homologous bone or cartilage or equivalent replacement materials. While autologous materials are not available indefinitely, infectiological considerations must be considered in homologous transplants.

The main problem of degenerative or traumatic joint disease is that the damaged articular cartilage has little ability to regenerate. It is known to treat such cartilage defects by means of autologous chondrocyte transplantation (ACT) as a biological therapy method. For the first time ever, the new formation of hyaline cartilage in a joint cartilage defect was detected with this therapy method.

The principle of this method is based on injecting in vitro cultured cartilage cells of the patient after debridement of the degenerated cartilage under a sewn on the defect periosteal flap (Peterson method). The process is technically demanding and raises methodological problems; For example, the long-term fate of cartilage cells in the defect is not guaranteed. Furthermore, it is unclear whether the cultured cartilage cells at the time of transplantation in the Are able to form the matrix needed for the permanent defect filling. The transplanted cells are mostly in a dedifferentiated state and have morphological and physiological similarities with fibroblasts.

From the DE 197 21 661 A1 It is known to use novel materials for the replacement of bone or cartilage, which are characterized by a specific structure of known materials. It is a bone or cartilage implant based on a three-dimensional grid, which consists essentially of a plurality of regularly arranged rods of a partially or completely bioresorbable material. The rods form a geometric three-dimensional structure, which structure is matched in terms of elasticity and strength to the tissue that is to replace it in the body of the patient. The rods may consist of poly-D-lactides, poly-L-lactides, poly-DL-lactides, hydroxyapatites, calcium phosphates or mixtures of these substances, which essentially contain calcium phosphates or hydroxylapatites, collagen, agar or gelatin.

The WO 99/08728 discloses an osteoinductive or chondroinductive mixture of factors embedded in nanospheres. Also disclosed is a system comprising a biodegradable matrix containing, for example, collagen type I or type II and embedded therein the factors enveloped by the nanospheres. The nanospheres are designed as polymer particles.

From the WO 95/33821 It is known to produce three-dimensional structures of collagen, wherein the interstitial areas are bridged by, for example, fibroblasts or chondrocytes and a cultivation of this network is carried out in a culture medium. This document describes the cultivation of said cells on this three-dimensional network as well as the use of this artificial tissue as a cartilage replacement.

The WO 98/17791 describes the isolation and use of precursor chondrocytes that can be methodically propagated in culture and used for the use of therapeutically useful cartilage tissue. Here, too, the cultivation of chondrocytes on a three-dimensional network is described, whereby the interstitial spaces are bridged by the chondrocytes.

The WO 99/00152 discloses a method for producing a bioartificial graft wherein from an allogeneic or xenogeneic tissue all antigen-reactive cells are removed by enzymatic or chemical treatment and the cell-free, non-denatured material so obtained is colonized with desired autologous cells, thereby producing a graft ready for immediate use is obtained.

In the known methods has the disadvantage that transplanted or cultured cartilage cells do not reach their original synthesis performance again.

The chondrocytes embedded in the intercellular substance of the cartilage namely the chondrocytes dedifferentiate under in vitro culture conditions during the cultivation period. While primary cartilage cells (P0 culture), after isolation from cartilage tissue, still show cartilage cell-typical synthesis lines, such as the formation of type II collagen type II and type XI, cultured cells lose these typical properties during further passages (P1-PX) in vitro.

In addition, cartilage cells, unlike other cells, are difficult to cultivate. Cultivated cartilage cells are in a dedifferentiated state and are morphologically and physiologically similar to fibroblasts. However, especially for larger defects, the proliferation of autologous chondrocytes by culture and passage is necessary for the production of sufficient quantities of transplantation material from small samples.

The dedifferentiation of the cartilage cells can hitherto only be prevented by the addition of additional growth stimulants, which makes the use of these cultures in the in vivo range more difficult since the growth stimulants can interact adversely with the immune system of the recipient organism. Furthermore, it is not ensured by the known methods that the cells maintain their natural or largely natural metabolism even after several passages during the cultivation.

In addition, the known three-dimensional structures for the cultivation of cartilage cells can not be specifically adapted to the particular cartilage defect in the previous methods. Furthermore, it is not ensured that the transplanted cartilage cells remain permanently and differentiated at the site of the defect, thus enabling the regeneration of the cartilage.

In fact, to date, there has been no transplantation procedure which ensures a permanent fate of transplanted cartilage cells at the site of the defect and associated cartilage regeneration.

Thus, the technical problem underlying the present invention is to provide cartilage grafts as well as methods and means for their production which allow improved treatment of cartilage diseases, in particular improved healing or regeneration of the treated cartilage defect.

The invention solves the underlying problem by providing means for redifferentiation and / or propagation of dedifferentiated cartilage cells, wherein the dedifferentiated cartilage cells are cultured in a three-dimensional gelatinous biomatrix where they can redifferentiate and resume their cell-typical metabolic activities.

The biomatrix according to the invention contains a collagen scaffold newly constituted from a, preferably fresh, collagen solution at a concentration of at least 1.5 mg collagen / ml biomatrix, preferably 1.5 to 4 mg collagen / ml biomatrix. This collagen scaffold is made of a preferably cell-free, acidic collagen solution, preferably having a pH of 0.1 to 6.9, preferably 2.0 to 5.0, preferably 3.0 to 4.5, in particular 3.2 to 4.2 and more preferably 3.8, which was at 2 to 10 ° C, preferably 4 ° C, prepared and stored. This collagen solution is to prepare a cell-free biomatrix at 2 to 10 ° C, preferably at 4 ° C, with a solution of medium, in particular a conventional cell culture medium, buffer, for example Hepes buffer, and serum, in particular human autologous serum, and gelled by raising the temperature to, for example, room temperature or 37 ° C. To prepare a cell-containing biomatrix, preferably precultured cells, for example cartilage cells or precursor cartilage cells, are introduced into the solution of medium, buffer and serum and then at 2 to 10 ° C., preferably 4 ° C., with likewise 2 to 10 ° C. , preferably 4 ° C, tempered collagen solution. The cells thus embedded in the biomatrix can then be cultured and, if appropriate, removed again. Subsequently, for example, gelled at room temperature or 37 ° C and the biomatrix covered with cell culture medium. The method according to the invention advantageously makes it possible to carry out intermediate cultivation of the cells in a biomatrix according to the invention, wherein the chondrocytes, for example the P2 culture, in the three-dimensional biomatrix are stimulated for the renewed synthesis of cell-typical matrix proteins, in particular collagen II, whereas in known cultures no collagen formation can be detected. Advantageously, the cells can then be removed again from the biomatrix and cultivated. By means of the redifferentiation of dedifferentiated cartilage cells made possible according to the invention, it is possible to cultivate and / or multiply chondrocytes over a relatively long period of time without the cells having the specific synthesis services necessary for the cartilage assembly to lose. Advantageously enough cell material can thus be made available for the production of cartilage grafts even if there is a small initial amount of cartilage tissue.

In the context of the present invention, the term "culturing of cells" is understood to mean maintaining, in vitro, the vital functions of cells in a suitable environment, for example by supplying and removing metabolic factors and products, and in particular also increasing them cells.

In the context of the present invention, cartilage cells or chondrocytes are understood as meaning naturally occurring or genetically engineered cartilage cells or their precursors, which may be of animal or human origin.

It is envisaged to cultivate differentiated cartilage cells, in particular while maintaining their differentiation, in a three-dimensional biomatrix as characterized above. Advantageously, the metabolic activities of the primary culture are obtained without dedifferentiation of the differentiated cartilage cells. Thus, the invention also provides a culture method for chondrocytes comprising suppression of redifferentiation.

It is envisaged that in their function, their morphology and / or their differentiation status to be examined cartilage cells are introduced into a said three-dimensional biomatrix, cultivated and thereby and / or checked thereafter. Advantageously, for example, the metabolism of the cartilage cells can be examined in vitro before the cartilage cells are used in vivo. The check may be, for example, the measurement of metabolic services as well as a morphological or functional check.

The invention also relates to screening and diagnostic methods wherein cultured cartilage cells according to the methods described above and thereby or subsequently examined, for example, on physiological, morphological and / or molecular biological parameters. In particular, degenerative or traumatic joint diseases or their absence can be detected. In the context of these methods, the effects of potential drugs and / or pathogens, antigens or the like on the cultured cells can also be investigated, for example in drug screening methods. In a preferred embodiment, the cells are cultured in the presence and absence of the agent to be investigated, and the observed effects are compared with one another.

In an advantageous embodiment, the cartilage cells are removed from the three-dimensional biomatrix following cultivation, for example by means of collagenase treatment and subsequent concentration, and further cultured in a conventional two-dimensional or three-dimensional cell culture. Advantageously, the advantages of two-dimensional and three-dimensional cultivation can thus be combined.

In a particularly advantageous embodiment, the cartilage cells are introduced into a three-dimensional biomatrix according to the invention and cultivated there in such a way that subsequently a transplantable cartilage replacement, also referred to as a cartilage graft, can be obtained. This cartilage replacement may be a joint cartilage replacement in a preferred embodiment. Advantageously, for example, during the phase of culturing the cells, the biomatrix can already be adapted to the shape of the cartilage defect.

An advantageous embodiment is an aforementioned method for producing a cartilage replacement, in particular articular cartilage replacement, wherein preferably the cartilage cells after culturing in the three-dimensional biomatrix dissolved out of this, preferably by means of collagenase treatment and centrifuging, and in-vorzugsweise-higher Cell density can be further cultured in a conventional two- or three-dimensional cell culture, whereby a transplantable cartilage replacement, in particular articular cartilage replacement, can be obtained.

The invention also relates to a cartilage replacement, in particular articular cartilage replacement, which has been produced by the process according to the invention and an optionally subsequent and / or preceding cultivation process of conventional type.

The invention also relates to the, preferably gelatinous, biomatrix in which the aforementioned culturing methods can be carried out, as a biomatrix without cartilage cells, as well as a biomatrix with cartilage cells. In the latter case, the combination of biomatrix and differentiated or redifferentiated cartilage cells cultured therein is also referred to as a cell-matrix-cartilage system or biograft. This biograft can be used directly for the treatment of cartilage diseases or defects.

According to the invention, the biomatrix is understood as meaning a gel structure comprising collagen, cell culture medium, serum and buffer, in particular Hepes buffer. The collagen solution used to prepare the biomatrix is a solution containing a high level of undenatured native collagen in acidic aqueous medium, especially at a pH of 0.1 to 6.9, preferably 2.0 to 5.0, in particular 3.0 to 4.5, preferably 3.2 to 4.2 and particularly preferably 3.8, for example in acetic acid, in particular 0.1% acetic acid solution. A high proportion is understood as meaning a proportion of undenatured collagen in the total collagen in solution of ≥ 50%, in particular ≥ 60, ≥ 70, ≥ 80, ≥ 90 or ≥ 95, in particular ≥ 99%. In a preferred embodiment, no lyophilized collagen is used. The collagen content of the solution is advantageously between 3 mg collagen / ml solution to 8 mg collagen / ml solution, preferably 6 mg collagen / ml solution. Advantageously, collagen is used in a preferred embodiment, after isolation from, for example Rat tails were incubated in 0.1% acetic acid for 3 to 14 days at 4 ° C with stirring and wherein undissolved collagen fractions were removed by centrifugation. As a cell culture medium, DMEM (Dulbecco's Modified Eagle Medium) is advantageously used. However, any other cell culture medium may be used which allows the cultivation of cartilage cells. As a serum, autologous human serum is advantageously used in a preferred embodiment, and as buffer, for example, Hepes. In a preferred embodiment, a 3 M concentration of Hepes is adjusted in this solution. The pH of the solution of cell culture medium, buffer and serum in a preferred embodiment is 7.5 to 8.5, for example 7.6 to 8.2, in particular 7.8. Of course, the biomatrix may also contain other factors such as growth factors, adhesives, antibiotics, selection agents or the like.

The invention therefore also relates to the process for preparing the biomatrix, wherein fresh collagen, for example from rat tails, is produced in a first step by collecting collagen fibers isolated from collagen-containing tissue in buffer solution, superficially disinfected in alcohol and then washed in buffer solution and then into a collagen acidic solution having a pH of 0.1 to 6.9, preferably 2.0 to 5.0, particularly preferably 3.0 to 4.0, in particular 3.3, for example, a 0.1% acetic acid solution, transferred become. Subsequently, in a further step, the collagen in the solution at 2 to 10 ° C, especially 4 ° C, for several days, for example, 3 to 14 days, stirred, the undissolved collagen fractions are centrifuged off and a finished collagen solution with a collagen content from 3 mg / ml to 8 mg / ml at 2 to 10 ° C, for example 4 ° C. Of course, it is possible to store the solution in a frozen state, for example at -10 ° C to -80 ° C, especially -20 ° C. In a third step, this collagen solution is treated with a solution having a pH of 7.5 to 8.5, preferably 7.6 to 8.2, in particular 7.8, containing double-concentrated cell culture medium, serum and buffer, in the ratio of preferably 1 : 1 mixed to obtain a biomatrix having a pH of from 7.0 to 7.8, preferably 7.4. To prepare a biograft, the solution of double-concentrated cell culture medium, serum and buffer is mixed with precultured and centrifuged cartilage cells, preferably 2 × 10 ⁴ to 2 × 10 ⁷ cells per ml, preferably 2 × 10 ⁶ cells. This solution of a pH of 7.5 to 8.5, preferably 7.6 to 8.2, in particular 7.8 is then in the ratio 1: 1 with the aforementioned collagen solution at 2 to 10 ° C, in particular 4 ° C. , mixed. The gel solution is then pipetted into culture vessels and, after gelling at 37 ° C., covered with medium. The biograft is then cultured for 3 to 8 days to be subsequently available for transplantation.

The biograft arrives in culture vessels in the operating room and can then be adapted by the physician by mechanical processing, for example with a knife, in size, thickness and shape to the defect. Subsequently, the biograft, for example by means of tissue adhesive, in particular fibrin glue, fixed in the defect. After about 5 minutes, the biograft is firmly anchored in the defect. The surgery can also be performed arthroscopically because the biograft is flexible.

Another use of the biotransplant is a combination of the biomatrix implant according to the invention with bone. The biograft is for example applied to autologous bone cylinders from the iliac crest, for example by means of fibrin glue. According to the invention, in particular the therapy of osteochondritis dissecans and defects on the femoral head in cerebral-paretic hip dislocation as well as the rehabilitation of traumatic and degenerative lesions in the knee joint is made possible. By early and causal therapy can be stopped according to the invention with the new method, a progression of traumatic or degenerative joint disease. For the treatment of osteoarthritis in younger people according to the invention is a unique and novel therapeutic method available, which significantly delays the endoprosthetic joint replacement, makes in individual cases superfluous. The therapy of inflammatory joint destruction is possible as an application of the method according to the invention.

The development of the gelatinous biomatrix according to the invention, in which autologous cells can be embedded in an exactly defined manner, makes it possible for the first time to transplant a defined amount of cartilage cells in a mechanically moldable and loadable matrix. The cell-matrix-cartilage system according to the invention, constructed from biomatrix and cartilage cells according to the invention, can be produced in any size and thickness and can be precisely adapted to the defect by mechanical processing. The biograft according to the invention also offers the advantage that, in contrast to ACT, an additional removal of the periosteum from the patient's tibia is omitted. By cultivating the in vitro cell-matrix-cartilage system of the present invention, it is possible to examine the cartilage cells before transplantation for their matrix production. This is an important prerequisite for the production of cartilage grafts, their testing for functionality and for quality assurance. It has thus been shown that the new synthesis of typical cartilage proteins, such as collagen type II, which are used for the primary stability of the transplant, are formed by the autologous cartilage cells in the matrix in vitro. According to the invention could be shown by experiments on the knee joint of minipigs that heal compared to the empty defect of the transplantation of empty matrix without cartilage cells or cartilage cell suspensions according to Peterson cell-matrix cartilage systems of the invention in the articular cartilage extremely well, completely fill the defect and are also pressure-stable ,

Finally, the invention also relates to a biomatrix for use in one of the aforementioned methods.

The invention also relates to methods for the treatment of cartilage diseases or defects, in particular degenerative, inflammatory and / or traumatic joint diseases, in which a cartilage replacement, biograft and / or cartilage cells produced according to the invention, into diseased or damaged tissue or cartilage - Used or bone areas, or is exchanged for the diseased or defective areas or will be.

The invention will be explained in more detail by means of examples.

Example 1: Collagen production

To make a collagen solution, collagenous tissue, such as tendons from rat tails, is used. All work is done under sterile conditions with sterile materials. The rat tails are then superficially disinfected after storage at -20 ° C for 5 minutes in 70% alcohol. Subsequently, the skin of the rat tails is peeled off and the individual collagen fibers are dissolved out. If other starting tissues are used, any cells present may be gently removed by mechanical, enzymatic or chemical treatment. The collagen fibers are collected in phosphate buffered saline (PBS) (pH 7.2), surface-disinfected in 70% alcohol for 10 minutes and then washed with PBS. The weight of the collagen fibers is determined and the fibers are transferred to a 0.1% acetic acid solution (about 8 to 12 mg / ml). For 3 to 14 days, this mixture is stirred at 4 ° C and then centrifuging the undissolved collagen parts (1000 rpm, 1 hour, 8 ° C). The finished collagen solution in a preferred embodiment has a collagen content of 3 mg / ml to 8 mg / ml. The collagen is thus present as a starting material, in a high proportion, as defined above, in solution rather than in fiber, framework or matrix form. Preferably, the resulting collagen solution is cell-free and made from fresh, non-lyophilized collagen.

Example 2: Preparation of biomatrix or biograft

Kollagenlösung:

min. 3 mg/ml in Essigsäure

Pufferlösung:

77,5 ml doppeltkonzentriertes Medium (zum-Beispiel DMEM)
20 ml Serum
2,5 ml Hepes-Lösung (3 mol/l, pH 7,8)

All work is done under sterile conditions with sterile materials.

Collagen solution:

minute 3 mg / ml in acetic acid

Buffer solution:

77.5 ml double concentrated medium (for example DMEM)
20 ml serum
2.5 ml Hepes solution (3 mol / l, pH 7.8)

Both solutions are stored at 4 ° C.

The biomatrix consists of a collagen solution in 0.1% acetic acid (collagen from rat tail with a collagen content of 3 mg / ml to 8 mg / ml, preferably 6 mg / ml) and a buffer solution of double-concentrated medium, serum and Hepes solution. Shortly after mixing these two components in the ratio of preferably 1: 1 gelled at temperatures above 4 ° C, for example, room temperature, a three-dimensional newly constituted collagen structure.

For the preparation of the biograft, ie a cell-containing biomatrix, precultivated and centrifuged in the usual way. (1000 rpm, 10 min., RT) ² × 10 ⁴ to ² × 10 ⁷ cartilage cells / ml in the buffer solution, preferably ² × 10 ⁶ , taken up, suspended and mixed with equal parts of the collagen solution at 4 ° C. This gel solution is pipetted into culture vessels and, after gelling at 37 ° C., is covered with medium to form a newly constituted collagen structure with cartilage cells embedded therein. The material is then available for transplantation after 3 to 8 days of culture.

If desired, the cells can be released again by collagenase treatment and subsequent centrifugation from the biomatrix and be won. The resulting differentiated cells may be the starting point for further cultivations.

Example 3: Transplantation at the open joint and arthroscopic application

Both in open-joint transplantation and in arthroscopic application, the cartilage matrix is initially mechanically adjusted in size and shape to the cartilage defect. Subsequently, the graft is introduced into the defect and fixed there by means of fibrin glue. Since the described cell-matrix cartilage system is on the one hand flexible but nevertheless dimensionally stable material, it is possible to virtually roll up this biograft in the tubular arthroscopy instruments and thus to introduce them into the defect. Due to their dimensional stability, the grafts return to their original shape at the destination and can now be fixed in place with fibrin glue.

Example 4: Results of PCR Studies

The following table reflects the results of PCR tests. The aim of these studies was to demonstrate that the cultivation according to the invention results in a redifferentiation of chondrocytes in a biomatrix according to the invention prepared according to Examples 1 and 2. From the table it can be seen that starting from a PO culture in the course of the subsequent conventionally performed passages P1 and P2, the ability of the chondrocytes to produce collagen type II is lost. The cells dedifferentiate in the course of this passage. In contrast, a P2 passage in a biomatrix according to the invention leads to the redifferentiation of dedifferentiated chondrocytes, which is evidenced by the recovered ability to produce collagen type II. <u> Table: </ u> Results of the PCR investigations: The RNA was isolated from the chondrocytes of the individual cultures and transcribed into cDNA. PO-2D P1-2D P2-2D P2 collagen 2x10 ⁶ / ml 3 weeks 2 weeks 3 weeks 3 weeks β2Microglobulin + + + + Collagen type I + + + + Collagen type II + - - + Collagen type XI + + + + Bmp 2 + + + + Bmp 4 + + + + Bmp 7 - - - -

• β2-Mikroglobulin ist ein konstitutiv exprimiertes Gen und dient als Positivkontrolle;
• Kollagen Typ II und XI sind knorpelspezifische Kollagen-Typen;
• Bone morphogenetic proteins (Bmp) 2,4 und 7 spielen eine Rolle bei der Differenzierung des Knorpelgewebes.

The expression (transcription) of chondrocyte-specific genes was tested by PCR amplification with suitable primers and subsequent gel electrophoresis:

• β2-microglobulin is a constitutively expressed gene and serves as a positive control;
• Collagen type II and XI are cartilage-specific collagen types;
• Bone morphogenetic proteins (Bmp) 2,4 and 7 play a role in the differentiation of cartilage tissue.

Claims (5)

1. Method for preparing a collagen biomatrix, comprising the steps of:

   dissecting of collagen fibres from rat tails;

   transferring said collagen fibres into an acetic acid solution having a pH of 3.0 to 4.0;

   incubating said collagen fibres in said acetic acid solution for 3 to 14 days at a temperature of 2 to 10°C;

   separating non-dissolved collagen parts such that a collagen solution having a collagen content of 3 mg/ml to 8 mg/ml is obtained; and

   mixing the obtained collagen solution with a buffer solution having a pH of 7.5 to 8.5 at a temperature of 2°C to 10°C and elevating the temperature for gelling such that a biomatrix with a collagen content of 1.5 to 4.0 mg per 1 ml biomatrix having a pH of 7.0 to 7.8 is obtained.
2. Method according to claim 1, wherein the collagen fibres are present in the acetic acid solution in a proportion of 8 to 12 mg of collagen fibres per 1 ml acetic acid solution.
3. Method according to claim 1 or 2, wherein the acetic acid solution is a 0.1 % acetic acid solution.
4. Collagen solution as prepared by means of the method according to one of the claims 1 to 3.
5. Biomatrix as prepared by means of the method according to one of the claims 1 to 4.